Targeting ubiquitin-independent proteasome with small molecule increases susceptibility in pan-KRAS–mutant cancer models
Shihui Shen, … , Lei Li, Huaiyu Yang
Shihui Shen, … , Lei Li, Huaiyu Yang
Published March 17, 2025
Citation Information: J Clin Invest. 2025;135(6):e185278. https://doi.org/10.1172/JCI185278.
View: Text | PDF
Research Article Oncology

Targeting ubiquitin-independent proteasome with small molecule increases susceptibility in pan-KRAS–mutant cancer models

Abstract

Despite advances in the development of direct KRAS inhibitors, KRAS-mutant cancers continue to exhibit resistance to the currently available therapies. Here, we identified REGγ as a mutant KRAS–associated factor that enhanced REGγ transcription through the KRAS intermediate NRF2, suggesting that the REGγ-proteasome is a potential target for pan-KRAS inhibitor development. We elucidated a mechanism involving the KRAS/NRF2/REGγ regulatory axis, which links activated KRAS to the ATP- and ubiquitin-independent proteasome. We subsequently developed RLY01, a REGγ-proteasome inhibitor that effectively suppressed tumor growth in KRAS-mutant cancer models and lung cancer organoids. Notably, the combination of RLY01 and the KRASG12C inhibitor AMG510 exhibited enhanced antitumor efficacy in KRASG12C cancer cells. Collectively, our data support the hypothesis that KRAS mutations enhance the capacity of the REGγ-proteasome by increasing REGγ expression, highlighting the potential of ubiquitin-independent proteasome inhibition as a therapeutic approach for pan-KRAS–mutant cancers.

Authors

Shihui Shen, Qiansen Zhang, Yuhan Wang, Hui Chen, Shuangming Gong, Yun Liu, Conghao Gai, Hansen Chen, Enhao Zhu, Bo Yang, Lin Liu, Siyuan Cao, Mengting Zhao, Wenjie Ren, Mengjuan Li, Zhuoya Peng, Lu Zhang, Shaoying Zhang, Juwen Shen, Bianhong Zhang, Patrick K.H. Lee, Kun Li, Lei Li, Huaiyu Yang

×

Figure 4

Identification of RLY01 as a potent REGγ-20S proteasome inhibitor.

Options: View larger image (or click on image) Download as PowerPoint
Identification of RLY01 as a potent REGγ-20S proteasome inhibitor. (A) A...
(A) A druggability pocket identified on the REGγ-20S complex. (B) Docking pose of the top 100 hits within the druggability pocket of 20S α6–α7 interface. (C) Schematic illustration of specific peptidase activity of the 11S proteasome in vitro. (D) The inhibition of 11S-activated (REGγ/α) 20S proteasome by RLY01 is peptide substrate-specific. Each value represents mean ± SEM (n = 3). ***P < 0.001, ****P < 0.0001; P values were measured by 1-way ANOVA with Tukey’s multiple-comparison test. (E) Chemical structure of RLY01. (F) Surface plasmon resonance sensor-grams and fits for the interaction between RLY01 and 20S proteasome. (G) The molecular docking model showing the interaction residues within the α6–α7 interface pocket of the 20S proteasome to RLY01. (H and I) Biotin pull-down assay. SW620 cell protein lysates were incubated with biotin, biotin-RLY01 (B-RLY01; 10 μM), B-RLY01 (10 μM) + 100 μM RLY01 (10× molar excess), B-RLY01 (10 μM) + 200 μM RLY01 (20× molar excess). (J and K) The interaction of 4 mutant variants (Y59A, N64A, R66A, L82A) of α7 and 4 mutant variants (Q146A, S150A, N152A, F154A) of α6 with RLY01. HA-α7, HA-α6, and 8 mutant variants were transiently expressed in HCT116 cells for 48 hours. Cell lysates were then incubated in vitro for 12 hours with biotin, 20 μM biotin-RLY01, or DMSO. Protein complexes were subsequently precipitated using streptavidin beads and analyzed by Western blot. (L) Scheme of DARTS. (M) The DARTS method was used for drug target identification. Immunoblotting shows that RLY01 protected the α7 subunit from pronase proteolysis. (N) Co-IP showing that RLY01 inhibits the interaction between REGγ and α7. FLAG-REGγ was ectopically expressed in HCT116-KO cells for 48 hours, and cell protein lysates were incubated with the indicated treatments for 12 hours in vitro, followed by detection by co-IP and Western blot analysis.